摘要
本研究以干旱荒漠盐生植物柽柳的幼嫩叶片为实验材料,探究摸索其总DNA的提取方法与RAPD-PCR的反应体系及扩增条件。结果表明,改良CTAB-高盐沉淀法较好地提取出了柽柳植物的总DNA,其产量、质量均有明显提高,比较适合于多数柽柳属植物的DNA提取;同时,通过单因素试验比较优化筛选出了适合于柽柳植物RAPD分析的最佳的PCR反应体系与扩增条件,其25μL的反应体系中包含有Mg2+=2.5 mmol/L、d NTPs=0.25 mmol/L、引物=0.30μmol/L、Taq DNA聚合酶=1.5 U、模板DNA=200 ng;其最适扩增条件为95℃预变性4 min,94℃变性30 s、36℃退火40 s、72℃延伸1 min、40个循环;最终在72℃下延伸10 min,4℃下保存;另外,通过6条多态性引物的扩增效果证实该优化体系比较稳定,扩增条件比较合适,能够满足多数柽柳植物分子多态性的检测要求。
In this research, the extraction method for total DNA from young leaves of Tamarix plants growing in arid, desert, saline-alkali habitat was expored and optimized their reaction system and amplification conditions of RAPD-PCR. The results showed that the total DNA extracted had good quality by using the improved method based on CTAB-high salt precipitation and its yield were also high significantly, so the method could be fit for the total DNA extraction of most plants in Tamarix L.. At the same time, by one-factor comparative experiments, the suitable RAPD-PCR system for Tamarix plants was developed as follows: in 25 μL PCR solution, including2.5 mmol/L Mg2+, 0.25 mmol/L d NTPs, 0.30 μmol/L primer, 1.5 U Taq DNA polymerase, 200 ng DNA template.Moreover, the amplification conditions were 95℃ for 4 min, 40 cycles of 94℃ for 30 s, 36℃ for 40 s, and 72℃for 1 min and a terminal extension step at 72℃ for 10 min, and then kept at 4℃. In addition, through the confirmatory test with six polymorphic primer pairs, the optimized reaction system could amplify stably and its amplification conditions can meet the requirement for molecular polymorphic analysis of most Tamarix L. plants.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2015年第9期2015-2023,共9页
Genomics and Applied Biology
基金
国家自然科学基金项目(31070194)资助
